ATUM uses two well characterized mammalian cell lines for bioproduction:
- HD-BIOP3 GS null CHOK1 cell line from Horizon Discovery
- DG44 from the lab of Dr. Lawrence Chasin (Columbia Univ), adapted to serum-free suspension
- Lineage traceability
- Host banks characterized (tested for mycoplasma, sterility, adventitious agents by PCR)
- Commercially available chemically defined media formulations.
Stable Expression on Transient Timelines
Generate High Expressing Stable Pools in 14 days
Timeline shown is applicable to HD-BIOP3 GS null CHOK1 cell line from Horizon Discovery co-transfected with ATUM’s transposase and transposons. Cell lines are available 12-14 weeks post transfection. Research cell banks (RCB) are released for clones that pass stability, 22-25 weeks post transfection.
Stable Pool Productivity
High productivity stable clones in ~12 weeks
HD-BIOP3 GS null CHOK1 cell line from Horizon Discovery was co-transfected with ATUM’s transposase and transposons with different vector and antibody combinations. Stable pools were established and productivity measured in non-optimized small scale shake flask or deep-well cultures.
Ranking of 165 clones
Clonal distribution in stable pools generated by Leap-In hyperactive transposase mRNA in a first-generation process. A transposon based antibody construct was co-transfected with transposase mRNA in HD-BIOP3 GS null CHOK1 cells from Horizon Discovery. Stable pools were established in glutamine-free conditions and the absence of MSX. Clones were isolated by limited dilution cloning in 96-well plates. Productivity of individual clones grown for 20 days in a 96-well plate with feeding was measured by Octet(R) (ForteBio).
8 clones selected for productivity assessment
The top 24 clones of 165 were further ranked based on productivity in a 7 day 24 deep well plate fed batch culture and the 8 most productive clones were selected. Productivity was measured in a non-optimized 14 day fed batch 125 ml shake flask culture. The specific productivity of clonal isolates was >40 pcd (picogram/cell /day).
pD2500 & pD3600 Stable Vectors
ATUM uses pD3600 vectors with glutamine synthase (GS), dihydrofolate reductase (DHFR) or puromycin selectable markers. A series of selectable marker expression levels are available. Lower levels of selectable marker expression produce a more stringent selection, which in turn results in higher expression of client proteins.
ATUM may clone client sequences into several different vectors to obtain maximal expression levels. Our stable expression vectors have combinations of promoters for expressing two, three or four open reading frames at controlled ratios. In some cases it may not be known what the desired expression ratios should be. ATUM can then create several different versions, and use pool productivity to guide us to the optimal configuration.
The pD3600 vectors are constructed in a modular way that allows easy modification of component elements. Versions are available that allow expression of up to four open reading frames in addition to the selectable marker.
ATUM vectors are transposons, which accelerates the generation of highly productive stable pools and lines.
ATUM has developed a set of tools for genomic integration of DNA constructs based on novel transposons and their cognate transposases. This system features several properties that make it particularly well suited to engineering mammalian cell lines for bioproduction:
- Integration sites are enriched in transcriptionally active chromatin
- Multiple copies of a transposon can be independently integrated into the genome of a single cell
- Transposition can be achieved in a broad range of hosts
- Transposon excision perfectly restores the original genomic sequence
- The entire sequence between two transposon ends is integrated into a host genome, and there is no payload size limit
Maintaining Structural Integrity
The main limitation in creating cell lines from multi-ORF constructs is what happens to the DNA when it gets into the cell. Typically DNA is randomly fragmented and those fragments are integrated, sometimes with additional rearrangements and concatemerization. This can undo all of the benefits of providing the genes in a single construct.
Structural integrity of the complete expression cassette is maintained in the presence of transposase as shown in the left panel. In the absence of transposase (right panel), structural integrity of the expression cassette is compromised as seen by truncations plus random concatemerization and scrambling of the expression cassette.
Transposases restore the benefit of carrying all genes on a single construct. Because the Leap-In transposases integrate the entire transposon into the expression host genome, they maintain the structural integrity of the construct, linkage of all genes to the selectable marker, and the desired expression balance.
pD2500 & pD3600 Stable Vectors
ATUM offers two stable vector series: the pD2500s and pD3600s, which share a similar structure. The pD2500s are compatible with one of our Leap-In transposases (LPN-1), but they do not require a transposase for good levels of expression. The pD3600s are compatible with both of our Leap-In transposases (LPN-1 and LPN-2), and we recommend that they only be used with a transposase. This is because we have deliberately attenuated the selectable markers in the pD3600s, so multiple integration events are required for cell viability under selection. The pD3600s are therefore capable of higher expression yields.
The pD2500 and pD3600 vectors are constructed in a modular way that allows easy modification of component elements. Versions are available that allow expression of up to four open reading frames in addition to the selectable marker.
Our stable vectors are built from several sets of mammalian functional elements:
- Metabolic selectable markers: Glutamine synthase (GS), dihydrofolate reductase (DHFR)
- Drug-resistance markers: puromycin, blasticidin, hygromycin, neomycin, zeocin
- Promoters: EF1a, CMV (murine and human), hybrids
- RNA processing elements: Introns, post-transcriptional response elements
- Insulators: Flanking the construct, and isolating transcriptional units from each other
The total number of possible combinations of these is very large, and we do not have all of these built. However construction is modular, so it is easy for us to create custom combinations if we don’t have the combination you want.
One Vector, Three or Four Open Reading Frames
Combinations of control elements can be used to create constructs carrying 3 or 4 open reading frames, enabling expression of all of the chains for a bispecific at controlled ratios.